1. Field of the Invention
This invention discloses multiple enzyme assays which measure the activity of at least one endogenous enzyme in a single aliquot of a sample or population of cells and multiple enzymes in a single aliquot of a sample provided that at least one of the enzymes is an endogenous enzyme. The present invention also discloses methods and kits for detecting the activity of multiple enzymes.
2. Background of the Prior Art
A wide variety of reporter gene assays are used in both biomedical and pharmaceutical research for the study of gene regulation and identification of cellular factors and chemical compounds that affect gene expression. Alam and Cook, Anal Biochem, 1990; 188:245-54; Bronstein, et al., Anal Biochem., 1994; 219:169-81. Reporter gene assays are useful in the study of gene regulatory elements because reporter gene activity, i.e., production of the reporter protein, is directly proportional to the transcriptional activity of the regulatory elements of the gene. A reporter gene construct for use in these assays contains one or more gene regulatory elements which are of interest, the minimal sequence requirements for transcription of a functional mRNA, and the coding sequence for a reporter protein. Alam. et al., Anal. Biochem., 188:245-254, 1990. Analysis of constructs containing various deletions within the regulatory region enables mapping of regulatory sequences necessary for transcription and cell specific expression.
Introduction of a reporter gene construct into cells, followed by quantitation of the expressed protein or its activity, provides an indirect measure of gene expression. For example, the sensitive quantitation of reporter gene products is important for analysis of gene expression, signal transduction pathways, identification of protein interactions, and drug discovery. Further, quantitation of the reporter gene enables mapping of the gene promoter and enhancer regions, analysis of gene expression mechanisms, and screening of chemical and natural product libraries for effectors of gene expression.
Chemiluminescent reporter gene assays combine high sensitivity with broad dynamic range, typically 6-7 orders of magnitude. Chemiluminescent 1,2-dioxetane substrates for several reporter enzymes, including xcex2-galactosidase (xcex2-Gal), xcex2-glucuronidase and alkaline phosphatase (AP) are used in highly sensitive assays. These chemiluminescent assays provide superior alternatives to traditional colorimetric, fluorescent, and radioisotopic detection methods. 1,2-dioxetane chemiluminescent substrates have also been used in a dual assay for luciferase and xcex2-galactosidase reporter enzymes. Enzymatic cleavage of each chemiluminescent substrate produces a destabilized dioxetane anion, which fragments and emits light.
Sensitive chemiluminescent assays, not limited to reporter gene assays, have been described using dioxetane substrates. Bronstein, U.S. Pat. No. 4,978,614, incorporated herein by reference. These dioxetane substrates emit visible light following,enzyme induced degradation. Enhancement of the chemiluminescent degradation of 1,2-dioxetanes by enhancer substances comprising certain water soluble molecules, such as globular proteins or synthetic polymers that have hydrophobic regions, has been described. Vovta et al., U.S. Pat. No. 5,145,772, incorporated herein by reference. These dioxetane substrates are also used in reporter gene assays for alkaline phosphatase, xcex2-galactosidase, and xcex2-glucuronidase. See e.g., Bronstein. et al., Anal. Biochem., 219:169-181, 1994, and citations therein. The use of dioxetane substrates and enhancers in reporter gene assays has been described in U.S. application Ser. No. 08/579,787, incorporated herein by reference. U.S. application Ser. No. 08/579,787 describes assays in which the products of multiple reporter genes are sequentially quantitated in the same aliquot of cell extract. Simple, rapid, and highly sensitive combined multiple reporter gene assays which detect commonly used reporter genes are described which do not use radioisotopes or require external light sources. These assays produce enhanced levels of light and therefore increase the dynamic range and sensitivity of the assay and enable the use of a wide variety of instruments.
1,2-dioxetane substrates have been incorporated into the GALACTO-LIGHT(trademark), GALACTO-LIGHT PLUS(trademark) and GALACTO-STAR(trademark) assay systems available from Tropix, Inc., for quantitation of xcex2-galactosidase reporter enzyme activity and have been used with mammalian cell cultures, tissue extracts, microinjected frog embryos, protozoan parasites, yeast and bacteria. Jain, et al., Anal Biochem., 1991, 199:119-24; Bronstein, et al. Bioluminescence and Chemiluminescence: Fundamental and Applied Aspects, (Campbell, et al., eds) Chichester:Wiley, 1994, 20-3; Martin, et al., Bioluminescence and Chemiluminescence: Molecular Reporting with Photons, (Hastings, et al., eds.), Chichester:Wiley, 1997, 525-8; Bronstein et al., Clin. Chem., 1996, 42:1542-6. The GUS-LIGHT(trademark) system is used for xcex2-glucuronidase reporter detection. Bronstein. et al., BioTechniques., 1994, 17:172-7. CSPD(copyright) substrate is utilized in the PHOSPHA-LIGHT(trademark) assay system for quantitation of either secreted or non-secreted forms of the human placental alkaline phosphatase (PLAP) reporter enzyme. Bronstein, et al., Bioluminescence and Chemiluminescence: Fundamental and Applied Aspects, (Campbell, et al., eds.) Chichester:Wiley, 1994, 20-3; Bronstein, et al., Clin. Chem., 1996, 42:1542-6; Bronstein, et al., BioTechniques, 1994, 17:172-7. The DUAL-LIGHT(copyright) system by Tropix, Inc. combines a 1,2-dioxetane with luciferin in a single-tube assay for xcex2-Gal and luciferase reporter enzymes. Martin, et al., BioTechniques, 1996, 21:520-4; Bronstein. et al., Bioluminescence and Chemiluminescence: Molecular Reporting with Photons, (Hastings, et al., eds.) Chichester:Wiley, 1997, 451-7.
Currently, multiple reporter gene assays are commonly used to provide controls for efficiency of transfection. In such assays, cells are transfected with a mixture of two separate plasmids, each having a different reporter gene. The expression of one reporter gene is controlled by different regulatory regions being studied while the other reporter gene, acting as a control, is generally constitutively expressed by a standard promoter or enhancer. The activity of the experimental reporter enzyme is normalized to the activity of the control reporter enzyme.
The measurement of multiple enzyme activities in a single assay provides several capabilities. Transfection normalization can be performed by quantitation of both experimental and control reporter enzymes. Pharmaceutical screening strategies benefit from multiple reporters to distinguish the effect of a compound on a specific transcription factor from a non-specific effect on gene expression, or for multiplex screening of several drug targets. These advantages are described in co-pending U.S. application Ser. No. 08/579,787, filed Dec. 28, 1995.
While reporter enzyme expression is useful for measuring gene regulation, it is also desirable to have a mechanism to measure cell number, cell adhesion, cytotoxicity, and cell proliferation. Reporter enzymes may have limited usefulness for performing these measurements because the promoter used for controlling such a reporter gene preferably acts independent of the exogenous compounds added to the cells for testing gene expression. One skilled in the art would need a gene construct that is expressed at a constant level by the cell regardless of what is added to the test cells. For example, one would have to use a reporter enzyme linked to a strong promoter that is not affected by the test compounds.
The measurement of both a reporter enzyme and endogenous cellular enzyme activity provides assays for normalization of reporter enzyme activity to cellular proteins, or potentially enabling simultaneous quantitation of the reporter activity and cell number, cell proliferation, cell adhesion, or cytotoxicity. It would be desirable to have a method for .measuring cell number, cell proliferation, cell adhesion, and cell health that does not require the use of a reporter enzyme to make such measurements.
Techniques for quantitating cell number to normalize or to measure cell proliferation, growth inhibition, cell adhesion or cytotoxic effects are presently known and include various methods for measurement of cellular enzyme activities, vital dye staining, and cellular metabolism. The necessity of testing a separate portion of the sample for measurement of reporter enzyme activity and for measurement of cell number decreases the precision of the assay and may introduce experimental errors into the results. Therefore, a multiple enzyme assay which is sequentially performed on the same aliquot of cell extract would simplify the assay procedure and minimize experimental errors. The use of multiple enzymes can improve the efficiency and information content of high throughput screening for drug discovery. It would be therefore useful to have a method for measuring these factors without requiring the testing of more than one single aliquot of a sample.
U.S. Pat. No. 5,744,320, to Sherf. et al. describes a dual-enzyme reporter system which measures two individual reporter enzymes produced by cells genetically manipulated to simultaneously express two different reporter enzymes. Sherf. et al., describe the use of dual luciferase reporter enzymes and the use of a quench and activate reagent for measuring the activity of those enzymes. However, Sherf et al. fail to describe a method that measures an endogenous enzyme.
Accordingly, it remains a goal to those of ordinary skill in the art to develop a method for measuring multiple enzymes, at least one of which is an endogenous enzyme, e.g., for measuring cell number, cell proliferation, cell adhesion or cytotoxicity in an assay. It is a further goal to those ordinarily skilled in the art to develop a method that measures at least one reporter enzyme and at least one endogenous enzyme in a single aliquot of a sample.
It is an object of the present invention to provide multiple enzyme assays which measure the activity of at least one endogenous enzyme in a single aliquot, thereby providing internal normalization for a cell number in a single sample.
It is another object of the invention to provide assays to measure the activity of multiple enzymes, wherein the multiple enzymes are at least one enzyme and at least one endogenous enzyme and wherein at least one enzyme is capable of reacting with a dioxetane. The use of 1,2-dioxetane substrates provide sensitive, versatile, and facile chemiluminescent assay systems for quantification of endogenous cellular enzymes.
It is yet another object of the present invention to provide a method of measuring the activity of multiple enzymes, wherein at least one enzyme is an endogenous enzyme, in an aliquot of cells. The method comprises quantifying the activity of a first enzyme by measuring the light signal produced by degradation of a first enzyme substrate by the first enzyme, and then quantifying the activity of a second enzyme by measuring the light signal produced by the degradation of a second substrate by the second enzyme, etc. All quantifications are performed on the same aliquot of cells extract.
The measurement of multiple enzyme activities in a single assay provides advantages. Measurement of both a reporter enzyme and an endogenous cellular enzyme activity is advantageous because it provides assays for normalization of reporter enzyme activity to cellular protein, or potentially enabling simultaneous quantitation of the reporter activity and cell number, cell proliferation, cell adhesion or cytotoxicity.
It is a further object of the present invention to provide kits for detecting the activity of multiple enzymes, at least one of which is an endogenous enzyme, in an aliquot of cells. The kit comprises the reagents for quantifying each of the enzymes, the substrates for each of the enzymes, wherein at least one of the substrates is a dioxetane, and optionally an accelerator solution containing a water soluble polymeric enhancer molecule.
The activity of the multiple enzymes can each be detected sequentially or simultaneously, depending on the instrumentation or detection device used.